CN116590380A - 一种抗基质干扰的病毒直裂裂解液 - Google Patents
一种抗基质干扰的病毒直裂裂解液 Download PDFInfo
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Abstract
本发明涉及一种抗样本基质干扰的病毒直裂裂解液，属于病毒检测技术领域。直接裂解液中包括具有如下浓度的组分：0.01～0.5MNaOH、0.01～0.5％(v/v)TritonX100、0.1～5％甲酰胺/DMSO、1～100mM硫酸铵、1～10mM亚精胺、0.01～1％(v/v)BSA、1～10％(v/v)甘油、10～100mMKCl、0.1～50mMEDTA/EGTA。克服了传统裂解法对下游反应的干扰，裂解产物无需提取、纯化。(1)裂解仅需2～10min，免提取，操作简便；(2)有效的抗基质干扰成分，裂解后产物无需纯化即可直接应用于等温扩增下游反应，不会造成反应抑制；(3)独特的酶稳定/增强成分组合，提升热稳定聚合酶在复杂干扰环境中的反应效率和特异性；(4)有效的核酸稳定成分，裂解后产物可长期保存，常温放置6h，4℃放置24h，‑80℃放置21天，不影响检出结果。
Description
技术领域
本发明涉及一种抗样本基质干扰的病毒直裂裂解液，属于病毒检测技术领域。
背景技术
目前，在病毒核酸检测中，通常需要对病毒样本进行裂解后的提取和纯化处理才可以进行扩增反应并进行检测，用于避免样本中的干扰性成分或者裂解后的杂质等对后续检测过程(如：PCR、qPCR等)或检测结果的影响。
对于不同类型的样本，其基质中大部分含有一些反应干扰物或抑制性成分，按其来源可分为内源性干扰物和外源性干扰物。内源性干扰物是指存在于样本自身中的组成成分，如：免疫球蛋白、蛋白酶、血红蛋白及其代谢产物、血红蛋白中的乳铁蛋白、肌红蛋白、脂类、黏蛋白、尿素、离子、胆盐、多糖等。外源性干扰物是指外部引入的抑制物或干扰性成分，如：肝素抗凝剂、纤维素和硝酸纤维素、手套滑石粉、标本容器或采样器材上含有的抑制物。这些干扰物的存在可以在不同程度上影响后续检测反应的结果。
传统上应对反应干扰物的解决方法是对样本进行核酸提取，适用于病毒核酸提取方法主要分为柱提取和磁珠提取两类，步骤上大致都分为裂解、结合、洗涤、洗脱，四个部分。但从成本和可操作性方面，均存在成本高、耗时长且操作繁琐的问题，限制了基于核酸的检测技术在病原快速检测领域的应用和推广。因此，开发一种能够对样本进行直接裂解、释放核酸并且可以使下游反应(如，PCR、qPCR、RT-qPCR、恒温扩增等)抗样本基质干扰的裂解液，用于核酸检测，成为目前亟待解决的问题。
Direct PCR技术是指无需对核酸进行分离提取，直接以拭子样本为对象，加入目标基因引物进行PCR反应。它的模板既包括传统的DNA模版，也包含RNA模版的逆转录PCR。Direct PCR技术不仅仅是直接对拭子样本进行常规定性PCR反应，还包括Real-Time qPCR反应，这就要求反应体系具备极强的抗样本基质干扰能力、抗背景荧光干扰能力。DirectPCR技术针对的拭子样本，只需要核酸模版的释放，并不对干扰PCR反应的蛋白、多糖、盐离子等进行针对性去除。这要求裂解液既可以高效裂解病毒，又可以和下游反应体系中的核酸聚合酶和PCR Mix兼容，同时可以保护其在复杂的基质干扰环境中的酶的活性和复制准确性。
发明内容
本发明的目的是：解决病毒裂解后应用在恒温扩增过程中由于裂解产物复杂导致下游过程受到干扰的问题。
本专利中所采用的直接裂解液中包括具有如下浓度的组分：0.01～0.5MNaOH、0.01～0.5％(v/v)TritonX100、0.1～50mM螯合剂；
还包括：酶稳定和增强成分，其中包含1～10％(v/v)甘油、10～100mMKCl，还包含0.1～5％的甲酰胺或DMSO中的一种或者两者的混合物。当为混合物时，重量比是指两者之和的总重量；
还包括：抗基质干扰成分组合，其中包含1～100mM硫酸铵、1～10mM亚精胺和0.01～1％(v/v)BSA(牛血清蛋白)。
所述的螯合剂是EDTA或EGTA中的一种或两者的混合。
恒温扩增检测方法，包括如下步骤：
病毒样本裂解：采用上述的裂解液，将拭子样本取样后，投入到裂解液中涡旋混匀，裂解；
并且由裂解得到的样本不经过处理直接进行恒温扩增检测。
采用一个拭子时，裂解液的用量200ul～3ml。
裂解时间2～10min。
恒温扩增检测中，反应程序：42℃，10min；4℃，维持温度。
有益效果
本裂解液中，其关键组分主要分为2个部分：
1)抗基质干扰成分组合：BSA、硫酸铵、亚精胺；
2)酶稳定/增强成分组合：甲酰胺/DMSO、甘油、KCl；
其可以避免下游反应受到影响的原理是：1)BSA、硫酸铵和亚精胺的特定浓度的组合，可以降低/中和PCR反应背景干扰，增强聚合酶的反应特异性。其中，BSA可以在一定程度上中和PCR反应干扰因子，NH4 +可以去除模板错配，亚精胺可以增强反应特异性；2)甲酰胺或和DMSO、甘油、KCl当中4种或者其中3种配方的特定浓度的组合，可以有效增强聚合酶反应效率，提高反应产物得量。其中，DMSO的主要作用是可以打开DNA二级结构，使酶容易延伸；甲酰胺可以促进引物-模板退火，降低DNA的变性温度；K+促进引物吸附，增强聚合酶反应效率；甘油增加反应稳定性、提高聚合酶反应产物得量。
克服了传统裂解法对下游反应的干扰，裂解产物无需提取、纯化。
(1)裂解仅需2～10min，免提取，操作简便；
(2)有效的抗基质干扰成分，裂解后产物无需纯化即可直接应用于等温扩增下游反应，不会造成反应抑制；
(3)独特的酶稳定/增强成分组合，提升热稳定聚合酶在复杂干扰环境中的反应效率和特异性；
(4)有效的核酸稳定成分，裂解后产物可长期保存，常温放置6h，4℃放置24h，-80℃放置21天，不影响检出结果。
附图说明
图1是本专利中的流程图。
图2是新型冠状病毒恒温扩增检测(双靶标)试纸条结果。
图3是不同裂解液分组新型冠状病毒恒温扩增检测(双靶标)试纸条结果。
具体实施方式
实施例1
本实施例用于说明对新型冠状病毒样本进行恒温扩增检测。
本实施例中病毒直裂裂解液由0.02M NaOH、0.01％(v/v)TritonX100、0.05％BSA、15mM硫酸铵、5mM亚精胺、0.1％甲酰胺、0.1％DMSO、0.5％(v/v)甘油、20mM KCl、0.1mMEGTA组成。
裂解液适用于口腔、口咽、鼻、鼻咽拭子的病毒快速裂解，在本实施例中采用咽拭子，将拭子样本取样后，投入到500ul裂解液中涡旋混匀，裂解10min，取裂解后产物直接进直接进行恒温扩增反应，采用专利申请号2023101240798中的基于新冠病毒N基因(靶标基因1)及ORF1a基因(靶标基因2)双靶标及人源内参RNase P基因设计的检测试剂。恒温扩增反应程序：42℃，10min；4℃，hold；体系：50μL，并运行。
上述的裂解试剂的成分经过改进后，提高其抗干扰能力效果，本发明还对组分进行了调整，并进行平行对照试验，分组如下：
表1不同功能组分(组合)的裂解液组成
样本裂解方法与实施例1相同，取裂解后产物直接进行恒温扩增反应，结果如图3。
通过分组1和分组2的对比可以看出，抗基质干扰组合(BSA、硫酸铵、亚精胺)，获得的试纸条显色条带更为清晰，这与其组分的抗反应干扰性有关。
通过分组2和分组3的对比可以看出，酶稳定增强组合(甲酰胺、甘油、KCl)可以通过增强酶反应效率和稳定性，进一步提升试纸条显色条带的亮度；
通过分组2、3和分组4的对比可以看出，同时含有抗基质干扰组合及酶稳定保护组合的裂解液配方(分组4)，所获得的试纸条显色条带最清晰。
综上，含有抗基质干扰组合和酶稳定保护组合的裂解液配方，对于后续恒温扩增反应的稳定更有利，试纸条显色结果更清晰。
Claims (6)
1.抗基质干扰的病毒直裂裂解液，包括具有如下浓度的组分：0.01～0.5M NaOH、0.01～0.5％(v/v)TritonX100、0.1～50mM螯合剂；
其特征在于，还包括：酶稳定和增强成分，其中包含1～10％(v/v)甘油、10～100mMKCl，还包含0.1～5％的甲酰胺或DMSO中的一种或者两者的混合物；
还包括：抗基质干扰成分组合，其中包含1～100mM硫酸铵、1～10mM亚精胺和0.01～1％(v/v)BSA。
2.根据权利要求1所述的抗基质干扰的病毒直裂裂解液，其特征在于，所述的螯合剂是EDTA或EGTA中的一种或两者的混合。
3.一种恒温扩增检测方法，包括如下步骤：病毒样本裂解：采用上述的裂解液，将拭子样本取样后，投入到裂解液中涡旋混匀，裂解；
并且由裂解得到的样本不经过处理直接进行恒温扩增检测。
4.根据权利要求3所述的恒温扩增检测方法，其特征在于，采用一个拭子时，裂解液的用量200ul～3ml。
5.根据权利要求3所述的恒温扩增检测方法，其特征在于，裂解时间2～10min。
6.根据权利要求3所述的恒温扩增检测方法，其特征在于，恒温扩增检测中，反应程序：42℃，10min；4℃，维持温度。
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